Follicular development in cryopreserved Common Wombat ovarian tissue xenografted to Nude rats

The Northern Hairy-nosed Wombat (Lasiorhinus krefftii) is a highly endangered marsupial species and every possible option for sustaining the species needs to be explored. One important approach may be the development of assisted reproductive technologies in the non-endangered Common Wombat (Vombatus ursinus) and Southern Hairy-nosed Wombat (Lasiorhinus latifrons) for application in breeding the Northern Hairy-nosed Wombat.In this study, it was examined whether cryopreserved Wombat ovarian tissue would develop following xenografting to immunologically deficient rats. Ovarian tissue was collected from Common Wombats (n = 3) and cryopreserved as small cortical pieces. After thawing the cortical pieces were grafted underneath the kidney capsule of Nude rats (n = 16). The grafts were recovered at 2, 4, and 10 weeks after transplantation and their gross and histological appearance investigated. Two weeks after grafting (n = 2), the tissue was revascularized and healthy primordial follicles were present. At week 4 (n = 2), some follicular development was present. At week 10, six rats received human chorionic gonadotrophin (hCG) to trigger follicle and oocyte maturation while another six rats were not given any treatment. The administration of hCG did not induce preovulatory follicles and oocyte maturation although type 5 follicles were present in ovarian tissue collected 10 weeks posttransplantation in both treated and untreated groups. This study demonstrates for the first time that Wombat ovarian tissue can survive and function when grafted into immunocompromized rats and that Wombat ovarian follicles can be recruited to growth and development in an ovarian xenograft. This model system has the potential to produce mature oocytes from endangered species for use in assisted reproductive technologies such as in vitro fertilization (IVF), intracytoplasmic sperm injection (ICSI), and mature oocytes from non-endangered species for nuclear transfer which may be necessary for the preservation of critically endangered species.     

The Association of Cortactin with Profilin-1 Is Critical for Smooth Muscle Contraction

Profilin-1 (Pfn-1) is an actin-regulatory protein that has a role in modulating smooth muscle contraction. However, the mechanisms that regulate Pfn-1 in smooth muscle are not fully understood. Here, stimulation with acetylcholine induced an increase in the association of the adapter protein cortactin with Pfn-1 in smooth muscle cells/tissues. Furthermore, disruption of the protein/protein interaction by a cell-permeable peptide (CTTN-I peptide) attenuated actin polymerization and smooth muscle contraction without affecting myosin light chain phosphorylation at Ser-19. Knockdown of cortactin by lentivirus-mediated RNAi also diminished actin polymerization and smooth muscle force development. However, cortactin knockdown did not affect myosin activation. In addition, cortactin phosphorylation has been implicated in nonmuscle cell migration. In this study, acetylcholine stimulation induced cortactin phosphorylation at Tyr-421 in smooth muscle cells. Phenylalanine substitution at this position impaired cortactin/Pfn-1 interaction in response to contractile activation. c-Abl is a tyrosine kinase that is necessary for actin dynamics and contraction in smooth muscle. Here, c-Abl silencing inhibited the agonist-induced cortactin phosphorylation and the association of cortactin with Pfn-1. Finally, treatment with CTTN-I peptide reduced airway resistance and smooth muscle hyperreactivity in a murine model of asthma. These results suggest that the interaction of cortactin with Pfn-1 plays a pivotal role in regulating actin dynamics, smooth muscle contraction, and airway hyperresponsiveness in asthma. The association of cortactin with Pfn-1 is regulated by c-Abl-mediated cortactin phosphorylation.     

Fluorescence lifetime imaging microscopy reveals sodium pump dimers in live cells

The sodium-potassium ATPase (Na/K-ATPase, NKA) establishes ion gradients that facilitate many physiological functions including action potentials and secondary transport processes. NKA comprises a catalytic subunit (alpha) that interacts closely with an essential subunit (beta) and regulatory transmembrane micropeptides called FXYD proteins. In the heart, a key modulatory partner is the FXYD protein phospholemman (PLM, FXYD1), but the stoichiometry of the alpha–beta–PLM regulatory complex is unknown. Here, we used fluorescence lifetime imaging and spectroscopy to investigate the structure, stoichiometry, and affinity of the NKA-regulatory complex. We observed a concentration-dependent binding of the subunits of NKA–PLM regulatory complex, with avid association of the alpha subunit with the essential beta subunit as well as lower affinity alpha–alpha and alpha–PLM interactions. These data provide the first evidence that, in intact live cells, the regulatory complex is composed of two alpha subunits associated with two beta subunits, decorated with two PLM regulatory subunits. Docking and molecular dynamics (MD) simulations generated a structural model of the complex that is consistent with our experimental observations. We propose that alpha–alpha subunit interactions support conformational coupling of the catalytic subunits, which may enhance NKA turnover rate. These observations provide insight into the pathophysiology of heart failure, wherein low NKA expression may be insufficient to support formation of the complete regulatory complex with the stoichiometry (alpha-beta-PLM)₂.     

Membrane topology of the Bcl-2 proto-oncogenic protein demonstrated in vitro

The Bcl-2 oncogenic protein was synthesized in vitro and shown to post-translationally integrate asymmetrically into microsomal membranes with no requirement for an amino-terminal signal sequence. Instead, a carboxyl-terminal hydrophobic domain of Bcl-2 served as an insertion sequence essential for membrane assembly since a Bcl-2 mutant lacking this domain completely lost its ability to associate with microsomal membranes. The data demonstrate that Bcl-2 is tightly associated with the lipid bilayer with the nature of an integral membrane protein. The membrane orientation of Bcl-2 was determined using a protease protection assay, which showed that it is predominantly localized to the cytoplasmic face of membranes. A similar type of membrane processing has been shown for cytochrome b5 and also suggested for the viral oncogenic protein polyoma middle-T antigen.     

Erythrocyte hemolysis by radiofrequency fields

A field-strength-dependent hemolytic effect of continuous-wave radiofrequency (RF) exposure in vitro has been demonstrated. Erythrocytes in whole heparinized rabbit blood were hemolyzed by a 2-h exposure to 50- or 100-MHz RF fields at field strengths of greater than 4 V/cm. An effect of comparable magnitude resulted from exposure to 10-MHz RF at a field strength of 9 V/cm. Sample temperatures were maintained at 22.5 degrees +/- 0.2 degrees C. There was no apparent involvement of heating or temperature gradients, nor were there any RF exposure effects on cellular K+ or Na+ concentration, nor on pH. The mechanism of the hemolytic effect is not known. Since the percentage of lysed erythrocytes was less than 1% and there was an absence of effects on cellular cation concentrations, RF radiation may have irreversibly altered the plasma membrane permeability of a sensitive subpopulation of red cells (possibly aged cells) leading to osmotic lysis. RF radiation at these frequencies appears to affect red cells in a manner that is qualitatively and quantitatively different from microwave radiation.     

Group B streptococci inhibit the chemotactic activity of the fifth component of complement

Infection with group B streptococci (GBS) is associated with a poor acute inflammatory response in which neutrophils fail to localize at the site of invasion. In the present studies, we have examined the effects of group B streptococci on C-derived chemotactic activity in human serum. Fresh human serum was activated to form C5a and C5adesarg by incubation with zymosan. The activated serum was then incubated with group B organisms, centrifuged, and the supernatants tested for chemotactic activity for human polymorphonuclear leukocytes. Group B organisms caused a dose-dependent decrease in C-dependent chemotactic activity. The degree of inhibition was profound with 1 X 10(9) bacteria/ml (10% of control). Experiments indicated that significant chemotactic factor inactivation occurred within 2 min of exposure to GBS organisms, while maximal inhibition occurred after 30 min incubation. A number of different strains of GBS of types I, II, and III possessed inhibitory activity. In contrast, group D streptococci, Staphylococcus aureus, Escherichia coli and Klebsiella pneumoniae failed to inhibit the C-derived chemotactic activity in human serum. Group A streptococci that were M protein positive also inactivated C-dependent chemotactic activity in serum, as previously reported. The inhibitory activity of the GBS strains could be abolished by heat or trypsin treatment but not by neuraminidase, pronase, or pepsin. C5a levels in zymosan-activated serum as measured by RIA were not decreased after incubation with an inhibitory strain suggesting that absorption was not involved. SDS-PAGE analysis revealed that group B streptococci degrade the C5a molecule, increasing its electrophoretic mobility by removing a fragment with a m.w. of approximately 650 Da. Thus, one of the reasons for the poor inflammatory response at the site of GBS infection may reside in the ability of these pathogens to inactivate C-derived inflammatory mediators. The GBS C5a-ase activity probably serves as an additional virulence factor for these organisms contributing to the poor inflammatory response characteristic of group B streptococcal infection.     

Identification of two type IIa IgG-binding proteins expressed by a single group A streptococcus.

Functional heterogeneity associated with Ig-binding proteins expressed by group A streptococci is well documented. In this study we have demonstrated that treatment of group A streptococcal isolate 64/14 with CNBr resulted in the solubilization of two different sized proteins that displayed identical functional reactivity with human IgG1, IgG2, and IgG4 (characteristics of a type IIa binding protein). Monospecific polyclonal antibodies to each form of type IIa molecule were prepared and no antigenic cross-reactivity between the two m.w. forms of type IIa binding protein could be detected. The smaller m.w. protein was shown to be identical or closely related to the recombinant type IIa protein cloned from strain CS110. These studies provide further evidence for the heterogeneity of type II Ig-binding proteins expressed by pathogenic group A streptococci.     

Studies on nuclear binding of dehydroepiandrosterone in hepatocytes.

Experiments were conducted to determine if dehydroepiandrosterone (DHEA) specifically binds to liver nuclei or interferes in the binding of other steroid hormones. Hepatocytes were isolated from obese, female Zucker rats that were treated with and without 0.6% DHEA in their diets for 2 weeks. The hepatocytes were incubated with either [3H]DHEA, [3H]estrone, or [3H]corticosterone. The nuclei isolated from the incubated cells from either control or DHEA-treated rats had no binding with [3H]DHEA, but had the expected binding with [3H]estrone and [3H]corticosterone. Furthermore, increasing concentrations of cold, unlabeled DHEA in the incubation media with [3H]estrone or [3H]corticosterone failed to have any effect on the binding of either of these steroid hormones to the nuclei. These results suggest that DHEA treatment does not exert its effects on cellular metabolism through a receptor-mediated mechanism like other steroid hormones or by interfering in the expression of steroid hormones such as estrone and corticosterone.